JP7522046B2 - Use of direct-fed microorganisms in preventing and/or treating E. coli-based infections in animals - Google Patents

Description

NRRL NRRL B-50510 NRRL NRRL B-50508 ATCC PTA-6507

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62/639,158, filed March 6, 2018, the disclosure of which is incorporated herein by reference in its entirety.

The technical field relates to the use of directly fed microorganisms in the prevention and/or treatment of animals with E. coli based infections.

E. coli is a gram-negative rod-shaped bacterium that normally inhabits the intestinal microflora or ecosystem of most mammalian and avian species. E. coli are classified into 150-200 serotypes or serogroups based on somatic (O), capsular (K), fimbrial (F), and flagellar (H) antigens. Most E. coli are commensals, i.e., they live in the intestine but do not harm the host organism. A small proportion of strains are pathogenic, producing virulence factors that cause disease. Some E. coli carry virulence genes in combinations not known to be associated with disease and may be considered potentially pathogenic. All E. coli may carry genes that confer resistance to antimicrobial agents.

In animals, pathogenic strains of E. coli are responsible for a variety of diseases, notably septicemia and diarrhea in newborn calves, acute mastitis in dairy cows, E. coli disease associated with chronic Mycoplasma respiratory disease, pericarditis, septic lung, and peritonitis in poultry, and Alabama rot in dogs.

E. coli is constantly shed into animals' immediate environment via feces, contaminating the cages, bedding, and flooring of indoor animals and the soil for outdoor animals. This E. coli can survive for long periods of time, in some cases for more than 10 weeks, and can spread via slurry and compost to fertilized fields and crops, and to ground and surface waters.

E. coli is transmitted to other animals via contaminated feed, handlers, and drinking water, and in some cases from farm to farm by vehicles such as transport trucks. Infection occurs in birds by the oral route or through inhalation of contaminated dust. E. coli from animals can also be transmitted to humans by direct contact, or by ingestion of contaminated food or water after spreading manure, or by ingestion of meat after contamination of carcasses at the slaughterhouse. Enteric infections caused by enterotoxigenic E. coli (ETEC) are the most common type of colibacillosis in young animals such as pigs and calves, typically manifesting as severe watery diarrhea. ETEC is also a significant cause of diarrhea among travelers ("traveler's diarrhea") and children in the developing world.

ETEC in pigs is often contagious, with identical strains being found in large numbers and in several sick pigs and from batch to batch. The strains are usually only shed for a few days after infection, probably due to the development of immunity.

The use of antibiotics in both human and animal treatment has given rise to antimicrobial resistance, which is now a major global threat to health. To address this global health concern, there is a drive to develop alternatives to antibiotics. There is therefore a need to discover novel and alternative approaches for the prevention and/or treatment of E. coli-based infections in animals.

In one embodiment, a composition for preventing and/or treating an E. coli based infection in an animal is disclosed, the composition comprising direct-fed microbial Bacillus-based components including Bacillus subtilis strain 3BP5 (NRRL B-50510); Bacillus amyloliquefaciens 918 (NRRL B-50508) and 15AP4 (PTA-6507).

In a second embodiment, the compositions disclosed herein may provide one or more performance benefits in an animal, the performance benefits being selected from the group consisting of increased weight gain, increased feed rate, improved gut barrier integrity, reduced mortality, and reduced fecal shedding of E. coli.

In a third embodiment, the directly administered microorganism is in the form of an endospore.

In a fourth embodiment, any of the compositions described herein further comprises at least one enzyme, which may optionally be encapsulated.

In a fifth embodiment, the at least one enzyme is selected from the group consisting of phytase, protease, amylase, xylanase, and beta-glucanase.

In a sixth embodiment, any of the compositions described herein may be a feed additive composition or a premix.

In a seventh embodiment, a feed comprising any of the feed additive compositions disclosed herein is disclosed.

In an eighth embodiment, a kit is disclosed that includes any of the feed additive compositions disclosed herein and instructions for administration.

In a ninth embodiment, a method of preventing and/or treating an E. coli based infection in an animal is disclosed comprising administering an effective amount of a composition comprising direct-fed microorganisms including Bacillus subtilis strain 3BP5 (NRRL B-50510); Bacillus amyloliquefaciens 918 (NRRL B-50508) and 15AP4 (PTA-6507). The composition so administered may provide one or more performance benefits in the animal, the performance benefits being selected from the group consisting of increased weight gain, increased feed rate, improved gut barrier integrity, reduced mortality, and reduced fecal shedding of E. coli. The composition may include any of the features described above or elsewhere in this disclosure.

FIG. 1 shows the effect of dietary treatments on tight junction protein expression as an indicator of gut health.

All cited patents, patent applications, and publications are incorporated herein by reference in their entirety.

A number of terms and abbreviations are used in this disclosure. The following definitions apply unless otherwise noted.

The articles "a," "an," and "the" preceding an element or component are intended to be non-restrictive with respect to the number of instances (i.e., occurrences) of that element or component. Thus, "a," "an," and "the" should be read to include one or at least one, and the singular form of an element or component also includes the plural, unless the number is clearly meant to be singular.

The term "comprising" refers to the presence of the features, integers, steps, or ingredients recited in the claim, but does not exclude the presence or addition of one or more other features, integers, steps, ingredients, or groups thereof. The term "comprising" is intended to include embodiments encompassed by the terms "consisting essentially of" and "consisting of." Similarly, the term "consisting essentially of" is intended to include embodiments encompassed by the term "consisting of."

When present, all ranges are inclusive and combinable. For example, if a range of "1 to 5" is listed, the listed range should be interpreted to include the ranges "1 to 4," "1 to 3," "1 to 2," "1 to 2 and 4 to 5," and "1 to 3 and 5," etc.

When used herein in connection with a numerical value, the term "about" refers to a range of ±0.5 of the numerical value, unless the term is specifically defined otherwise in the context. For example, the phrase "a pH value of about 6" refers to a pH value of 5.5 to 6.5, unless the pH value is specifically defined otherwise.

Every numerical upper limit given throughout this specification is intended to include every lower numerical limit, as if such lower numerical limit were expressly stated herein.Every numerical lower limit given throughout this specification will include every higher numerical limit, as if such higher numerical limit were expressly stated herein.Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly stated herein.

The terms "animal" and "subject" are used interchangeably herein. Animals include all non-ruminant (e.g., humans) and ruminant animals. In certain embodiments, animals are non-ruminant animals, such as horses and monogastric animals. Examples of monogastric animals include, but are not limited to, pigs and swine, such as piglets, growing pigs, and sows; poultry, such as turkeys, ducks, chickens, broiler chickens, and laying hens; fish, such as salmon, trout, tilapia, catfish, and carp; and crustaceans, such as shrimp and prawns. In further embodiments, the animal may be multigastric, such as a ruminant (e.g., but not limited to, cattle, heifers, goats, sheep, giraffes, bison, moose, elk, yaks, buffalo, deer, camels, alpacas, llamas, antelopes, pronghorns, and nilgais).

The term "ruminant" as used herein refers to a mammal that may obtain nutrients from plant-based foods by fermenting the food in a specialized stomach prior to digestion, primarily through microbial action. This process typically requires regurgitation of the fermented ingestion (known as cud chewing) and then re-chewing. The process of re-chewing the cud to further break down the plant material and stimulate digestion is called rumination. There are approximately 150 species of ruminant animals, including both domestic and wild species. Ruminant animals include, but are not limited to, cattle, cows, goats, sheep, giraffes, yaks, deer, elk, antelope, and buffalo.

The term "CFU" as used herein means "colony forming unit" and is a measure of viable cells where a colony represents an aggregate of cells derived from a single progenitor cell.

The term "direct-fed microorganisms" ("DFM"), as used herein, refers to a source of naturally occurring live (viable) microorganisms. DFM can include one or more of such naturally occurring microorganisms (e.g., bacterial strains). Categories of DFM include spore-forming bacteria (e.g., Bacillus and Clostridium) and non-spore-forming bacteria (e.g., lactic acid bacteria, yeast, and fungi). Thus, the term DFM encompasses one or more of the following: direct-fed bacteria, direct-fed yeast, direct-fed yeast or fungi, and combinations thereof.

Bacillus is a unique gram-positive rod-shaped bacterium that forms spores that are very stable and can withstand environmental conditions such as heat, humidity, and pH ranges. When ingested by animals, the spores germinate into active vegetative cells that can be used in food and pelleted feed.

The term "Bacillus-based ingredient" as used herein refers to (i) a Bacillus-based direct-fed microorganism, including a Bacillus bacterial strain described herein, (ii) a supernatant obtained from a Bacillus culture made from the strain, or (iii) a combination of (i) and (ii).

"Feed" and "food" mean any natural or artificial conventional feed, food, or component of such food, intended or suitable to be eaten, ingested, or digested by non-human animals and humans, respectively.

As used herein, the term "food" is used broadly to include human foods and food products, as well as foods (i.e., feed) for non-human animals.

The term "feed" is used in reference to products fed to animals in livestock farming. The terms "feed" and "animal feed" are used interchangeably. In a preferred embodiment, the food or feed is for consumption by monogastric and polygastric animals.

The term "probiotics" as used herein is defined as live microorganisms (including, for example, bacteria or yeasts) that, when ingested or applied topically in sufficient numbers, beneficially affect a host organism (i.e., by conferring one or more positive health benefits to the host organism). Probiotics may improve the microbial balance at one or more mucosal surfaces. For example, the mucosal surface may be the intestine, the urinary tract, the respiratory tract, or the skin. The term "probiotics" as used herein also includes live microorganisms that may stimulate beneficial branches of the immune system and simultaneously reduce inflammatory responses at mucosal surfaces (e.g., the intestine). There is no lower or upper limit for probiotic intake, but it has been suggested that a daily dose of at least ¹⁰ to ^{10 cfu} , preferably at least ¹⁰ to ¹⁰ cfu, and preferably ¹⁰ to ¹⁰ cfu, is effective to obtain beneficial health effects in a subject.

The term "prebiotic" refers to a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of beneficial bacteria.

The term "pathogen" as used herein means any causative agent of disease. Such causative agents may include, but are not limited to, bacterial causative agents, viral causative agents, fungal causative agents, and the like.

The term "E. coli-based infection" means a disease or infection, such as diarrhea, caused by E. coli.

The terms "derived from" and "obtained from" refer not only to proteins produced or producible by the strain of organism in question, but also to proteins encoded by DNA sequences isolated from such strains and produced in a host organism containing such DNA sequences. In addition, the terms refer to proteins encoded by DNA sequences of synthetic and/or cDNA origin and having identifiable characteristics of the protein in question.

The term "effective amount" means an amount of a particular ingredient administered to an animal sufficient to achieve a desired effect.

In one embodiment, a composition for preventing and/or treating an E. coli based infection in an animal is disclosed, the composition comprising a direct-fed microbial Bacillus-based component, including Bacillus subtilis strain 3BP5 (NRRL B-50510); Bacillus amyloliquefaciens strain 918 (NRRL B-50508) and strain 15AP4 (PTA-6507).

In a second embodiment, the compositions disclosed herein may provide one or more performance benefits in an animal, the performance benefits being selected from the group consisting of increased weight gain, increased feed rate, improved gut barrier integrity, and reduced fecal shedding of E. coli.

The Bacillus-based DFM components described herein may include live bacteria, may include supernatant, or may be a combination of live bacteria and culture supernatant. The preferred Bacillus-based DFM components are live bacteria.

In one embodiment, the DFM may be a spore-forming bacterial strain, and thus the term DFM may consist of or include spores (e.g., bacterial spores). Thus, the term "viable bacteria" as used herein may include bacterial spores (e.g., endospores or conidia). Alternatively, the DFM in the feed additive compositions described herein may not consist of or include bacterial spores (e.g., endospores or conidia).

The Bacillus-based DFM components described herein are combinations of the following strains:
Bacillus subtilis 3BP5 strain Accession No. NRRL B-50510,
Bacillus amyloliquefaciens strain 918 ATCC accession number NRRL B-50508, and Bacillus amyloliquefaciens strain 1013 ATCC accession number NRRL B-50509.

Strains 3BP5 and 1013 are described in International Publication WO 2013-329013, published on February 28, 2013.

The 15AP4 strain is described in U.S. Patent Application Publication No. 2005-0255092, published November 17, 2005.

In some embodiments, it is important that the Bacillus-based DFM component is heat tolerant, i.e., thermotolerant, such as from sporulation. This is especially true if the feed is pelleted. Bacilli can form stable endospores when growth conditions are unfavorable, and are very resistant to heat, pH, moisture, and disinfectants. If the bacteria/DFM is not a spore former, it should be protected to survive feed processing, as explained below.

The Bacillus-based ingredients described herein may be prepared as cultures and carriers (if used) and mixed in a ribbon or paddle mixer for about 15 minutes, although this time may be increased or decreased. The ingredients are blended to obtain a homogenous mixture of cultures and carriers. The final product is preferably a dry, free-flowing powder. Thus, the Bacillus-based ingredients may include a: Bacillus-based direct-fed microorganism comprising the three Bacillus bacterial strains described herein, or a supernatant obtained from a Bacillus culture, or a combination of both one or more Bacillus bacterial strains and a supernatant. Such Bacillus-based ingredients may then be added to an animal feed or feed premix. This Bacillus-based ingredient can be added on top of the animal feed ("top-feed") or can be added to liquids such as the animal's drinking water.

The inclusion of individual strains in the Bacillus-based DFMs described herein may be in percentages varying from 1% to 99%, preferably in percentages varying from 25% to 75%.

Suitable dosages of the Bacillus-based ingredients described herein in animal feed may range from about 1×10 ³ CFU/g feed to about 1×10 ¹⁰ CFU/g feed, preferably from about 1×10 ⁴ CFU/g feed to about 1×10 ⁸ CFU/g feed, and preferably from about 7.5×10 ⁴ CFU/g feed to about 1×10 ⁷ CFU/g feed.

Those skilled in the art will be readily aware of specific species and/or strains of microorganisms within the genera described herein that are used in the food and/or agricultural industries and are generally considered suitable for animal consumption. Animal feed may include plant materials such as corn, wheat, sorghum, soybean, canola, sunflower, or mixtures of any of these plant materials or plant protein sources for poultry, swine, ruminants, aquaculture, and pets.

The terms "animal feed", "feed" and "feedstuff" are used interchangeably and may include one or more feed ingredients selected from the group including: a) grains, such as small grains (e.g., wheat, barley, rye, oats, and combinations thereof), and/or large grains (e.g., corn or sorghum); b) grain by-products, such as corn gluten meal, distillers dried grains with solubles, and/or corn sorghum; c) cereal by-products, such as corn gluten meal, distillers dried grains with solubles, and/or corn sorghum; Solubles (DDGS) (especially corn-based distillers dried grains with solubles (cDDGS), wheat bran, wheat middlings, wheat shorts, rice bran, rice hulls, oat hulls, palm kernel, and citrus pulp; c) proteins from sources such as soybean, sunflower, peanut, lupin, pea, broad bean, cotton, canola, fish meal, dried plasma protein, meat and bone meal, potato protein, whey, copra, sesame; d) fats and oils from vegetable and animal sources; and/or e) minerals and vitamins.

When used as or in the preparation of a feed, such as a functional feed, the Bacillus-based ingredients described herein may be combined with one or more of the following: a nutritionally acceptable carrier, a nutritionally acceptable diluent, a nutritionally acceptable additive, a nutritionally acceptable adjuvant, a nutritionally active ingredient, such as at least one ingredient selected from the group consisting of proteins, peptides, sucrose, lactose, sorbitol, glycerol, propylene glycol, sodium chloride, sodium sulfate, sodium acetate, sodium citrate, sodium formate, sodium sorbate, potassium chloride, potassium sulfate, potassium acetate, potassium citrate, potassium formate, potassium acetate, potassium sorbate, magnesium chloride, magnesium sulfate, magnesium acetate, magnesium citrate, magnesium formate, magnesium sorbate, sodium metabisulfite, methylparaben, and propylparaben.

In a preferred embodiment, the Bacillus-based ingredient described herein may be mixed with a feed ingredient to form a feed. The term "feed ingredient" as used herein means all or a portion of the feed. A portion of the feed may mean one component of the feed or may mean multiple components of the feed (e.g., two, or three, or four, or more). In one embodiment, the term "feed ingredient" encompasses a premix or premix ingredients. Preferably, the feed may be forage or a premix thereof, a compound feed or a premix thereof. A feed additive composition comprising the Bacillus-based ingredient described herein may be mixed with a compound feed, mixed with a premix of a compound feed, mixed with forage, mixed with forage ingredients, or mixed with a premix of forage.

The term fodder, as used herein, means any food that is given to animals (as opposed to food that the animals must find for themselves). Fodder includes plants that have been cut.

The term fodder includes hay, straw, silage, compressed and pelleted feed, oil and mixed feed, including germinated grains and legumes.

The fodder may be obtained from one or more of the following plants: alfalfa (lucerne), barley, lotus grass, oilseed rape, cabbage (Chau moellier), kale, rapeseed (canola), rutabaga (Swedish turnip), tulip, clover, alsaike clover, red clover, subterranean clover, white clover, grasses, false oat grass, fescue, burgrass, brome grass, heath grass, and the like. grass), pastures (naturally mixed pastures, grassland-derived, orchardgrass, ryegrass, timothy grass, corn (maize), millet, oats, sorghum, soybeans, trees (tree shoots cut for tree-hay), wheat, and legumes.

The term "compound feed" means commercially available feed in the form of meal, pellets, nuts, cakes or grinds. Compound feed may be blended from various raw materials and additives. The blend is formulated according to the specific requirements of the target animal.

Formulated feeds may be complete feeds providing all of the nutrients required daily, they may be concentrates providing a portion of the feed (protein, energy), or they may be supplements providing only additional micronutrients such as minerals and vitamins.

The main ingredient used in compound feed is feed grains, which include corn, soybeans, sorghum, oats, and barley.

Premixes as referred to herein may suitably be compositions comprised of minor ingredients (e.g., vitamins, minerals, chemical preservatives, antibiotics, fermentation products, and other essential ingredients). Premixes are typically compositions suitable for blending into commercial feeds.

Any of the feeds described herein may include one or more feed ingredients selected from the group including: a) cereals, such as small grains (e.g., wheat, barley, rye, oats, and combinations thereof) and/or large grains (e.g., corn, sorghum); b) cereal by-products, such as corn gluten meal, distillers dried grains with solubles (DDGS), wheat bran, wheat middlings, wheat shorts, rice bran, rice hulls, oat hulls, palm kernels, and citrus pulp; c) proteins from sources such as soybean, sunflower, peanut, lupin, pea, broad bean, cotton, canola, fish meal, dried plasma protein, meat and bone meal, potato protein, whey, copra, sesame, and the like; d) fats and oils from plant and animal sources; e) minerals and vitamins.

Furthermore, such feed may comprise at least 30%, at least 40%, at least 50%, or at least 60% by weight of corn and soybean meal, or corn and full fat soybean, or wheat meal or sunflower meal.

Additionally or alternatively, the feed may include at least one high fiber feed ingredient and/or at least one by-product of at least one high fiber feed ingredient to provide a high fiber feed. Examples of high fiber feed ingredients include: wheat, barley, rye, oats, by-products from cereals such as corn gluten meal, distillers dried grains with solubles (DDGS), wheat bran, wheat middlings, wheat shorts, rice bran, rice husk, oat husk, palm kernel, and citrus pulp. Some protein sources may also be considered high fiber: proteins obtained from sources such as sunflower, lupin, broad bean, and cotton.

As described herein, the feed may be one or more of the following: formulated feeds and premixes, e.g., pellets, nuts, or (bovine) cakes; crops or crop residues: corn, soybean, sorghum, oats, barley, corn stover, copra, straw, rice husk, sugar beet waste; fish meal; freshly cut grass and other forage plants; meat and bone meal; molasses; oil cake and press cake; oligosaccharides; preserved forage plants: hay and silage; seaweed; seeds and grains (whole or prepared by crushing, grinding, etc.); germinated grains and legumes; yeast extract.

The term feed, as used herein, also encompasses pet food in some embodiments. Pet food is plant or animal material intended for consumption by pets, such as dog food or cat food. Pet foods, such as dog food and cat food, may be in dry form, such as kibble for dogs, or in wet canned form. Cat food may include the amino acid taurine.

The term feed may also encompass fish food in some embodiments. Fish food typically contains the macronutrients, trace elements, and vitamins necessary to keep the fish in captivity in good health. Fish food may be in the form of flakes, pellets, or tablets. Pelleted forms, some of which sink quickly, are often used for larger fish or bottom-dwelling species. Some fish food also contain additives such as beta-carotene or sex hormones to artificially enhance the coloration of ornamental fish.

Similarly, the term "feed" includes bird food, including both food used in bird feeders and food used to feed pet birds. Typically, bird food contains a variety of seeds, but may also include suet (beef or mutton fat).

As used herein, the term "contacted" refers to the indirect or direct application of the feed additive composition to a product (e.g., feed). Examples of application methods that may be used include, but are not limited to, treating the product with a material that includes the feed additive composition, direct application by mixing the feed additive composition with the product, spraying the feed additive composition onto the product surface, or immersing the product in a preparation of the feed additive composition.

The Bacillus-based ingredient may preferably be mixed with the product (e.g., feed). Alternatively, the Bacillus-based ingredient may be included in the feed emulsion or ingredients.

In some applications, it is important to have the Bacillus-based ingredient available on or at the surface of the product being affected/treated.

The Bacillus-based ingredient may be applied to dust, coat, and/or impregnate a product (e.g., a feed or feed ingredient) with a controlled amount of the Bacillus-based ingredient.

The DFMs described herein may be added at any suitable concentration, for example, at a concentration in the final feed product that provides a daily dose of from about 2×10 ³ CFU/g feed to about 2×10 ¹¹ CFU/g feed, preferably from about 2×10 ⁶ to about 1×10 ¹⁰ , preferably from about 3.75×10 ⁷ CFU/g feed to about 1×10 ¹⁰ CFU/g feed.

Preferably, the Bacillus-based ingredient will be thermally stable to heat treatment up to about 70° C.; up to about 85° C.; or up to about 95° C. The heat treatment may be carried out for about 30 seconds to several minutes. The term "thermally stable" means that at least about 50% of the Bacillus-based ingredient that was present/active before heating to a particular temperature is still present/active after cooling to room temperature. In a particularly preferred embodiment, the Bacillus-based ingredient is homogenized to produce a powder.

Alternatively, the Bacillus-based ingredient is formulated into granules (referred to as TPT granules) as described in WO 2007/044968, which is incorporated herein by reference.

In another preferred embodiment, when the feed additive composition is formulated into a granule, the granule comprises a hydration barrier salt coated onto the protein core. The advantages of such a salt coating are improved heat resistance, improved storage stability, and protection against at least one protease, including one or more bacterial strains, and/or other feed additives that adversely affect the DFM. Preferably, the salt used in the salt coating has a water activity of greater than _0.25 or a moisture content of greater than 60% at 20°C. Preferably, the salt coating comprises _Na2SO4 .

Feeds containing Bacillus-based ingredients can be manufactured using a feed pelleting process. Optionally, the pelleting process can include a steam treatment or conditioning step prior to the formation of pellets. The mixture containing the powder can be placed in a conditioner (e.g., a mixer with steam injection). The mixture is heated in the conditioner to a specific temperature such as 60-100°C, with typical temperatures being 70°C, 80°C, 85°C, 90°C, or 95°C. Residence times can vary from a few seconds to a few minutes or even hours. For example, 5 seconds, 10 seconds, 15 seconds, 30 seconds, 1 minute 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, and 1 hour.

For granules, the at least one coating may include a moisture hydrating material that accounts for at least 55% w/w of the granule; and/or the at least one coating may include two coatings. The two coatings may be a moisture hydrating coating and a moisture barrier coating. In some embodiments, the moisture hydrating coating may be 25% to 60% w/w of the granule and the moisture barrier coating may be 2% to 15% w/w of the granule. The moisture hydrating coating may be selected from inorganic salts, sucrose, starch, and maltodextrin, and the moisture barrier coating may be selected from polymers, gums, whey, and starch.

The granules may be produced using a feed pelleting process, and the feed pretreatment process may be carried out at 70°C to 95°C (e.g. 85°C to 95°C) for up to several minutes.

The Bacillus-based ingredient can be formulated into a granule for animal feed having a water activity of less than 0.5 prior to the steam-heated pelleting process, the granule comprising: a core; an active agent in the granule that retains at least 80% activity after storage and after the steam-heated pelleting process in which the granule is an ingredient; a moisture barrier coating; and a moisture hydrating coating that is at least 25% w/w of the granule.

The granules may have a moisture barrier coating selected from polymers and gums, and the moisture hydrating material may be an inorganic salt. The moisture hydrating coating may be 25% to 45% w/w of the granule, and the moisture barrier coating may be 2% to 10% w/w of the granule.

Granules can be produced using a steam-heated pelletizing process that can be carried out at 85°C to 95°C for up to several minutes.

Alternatively, the composition is in a liquid formulation suitable for consumption, preferably such liquid consumables include one or more of a buffer, a salt, sorbitol, and/or glycerol.

The feed additive composition may also be formulated by applying (e.g., spraying) the Bacillus-based component onto a carrier substrate, such as ground wheat.

In one embodiment, such feed additive compositions including the Bacillus-based components described herein may be formulated as a premix. By way of example only, the premix may include one or more feed ingredients, such as one or more minerals and/or one or more vitamins.

Alternatively, the composition is in a liquid formulation suitable for consumption, preferably such liquid consumables include one or more of a buffer, a salt, sorbitol, and/or glycerol.

The feed additive composition may also be formulated by applying (e.g., spraying) the Bacillus-based ingredient onto a carrier substrate, such as ground wheat.

In one embodiment, such Bacillus-based ingredients described herein may be formulated as a premix. By way of example only, the premix may include one or more feed ingredients, such as one or more minerals and/or one or more vitamins.

It will be appreciated that the Bacillus-based ingredients disclosed herein are suitable for addition to any suitable feed material.

As used herein, the term feed material refers to the basic feed material consumed by an animal. It will be further understood that the feed material may include, for example, at least one or more unprocessed grains, and/or processed plant and/or animal materials (e.g., soybean meal or bone meal).

It will be understood by those skilled in the art that different animals have different feed requirements, and that the same animal may have different feed requirements depending on the purpose for which the animal is being raised.

Preferably, the feed may include feed materials containing protein-rich ingredients such as maize or corn, wheat, barley, triticale, rye, rice, tapioca, sorghum, and/or by-products, as well as soybean meal, rapeseed meal, canola meal, cottonseed meal, sunflower seed meal, animal by-product meal, and mixtures thereof. More preferably, the feed may include animal fat and/or vegetable oil.

Optionally, the feed may also include additional minerals, such as calcium, and/or additional vitamins. Preferably, the feed is a corn soybean meal mix.

In another aspect, a method of producing a feed is provided. The feed is typically produced in a feed mill, where the raw material is first ground to a suitable particle size and then mixed with appropriate additives. The feed may then be produced as a mash or pellets; the latter typically involves raising the temperature to a target level and then passing the feed through a die to produce pellets of a particular size. The pellets are cooled. Liquid additives such as fats and enzymes may then be added. The production of the feed may also include further steps including extrusion or expansion prior to pelleting, particularly by suitable techniques which may include at least the use of steam.

The feed can be for monogastric animals such as poultry (e.g., broilers, layers, broiler breeders, turkeys, ducks, geese, waterfowl), pigs (all age categories), pets (e.g., dogs, cats), or fish; preferably, the feed is for poultry.

The Bacillus-based ingredients described herein can be placed on top of, or top-fed to, an animal's feed. Alternatively, the Bacillus-based ingredients described herein can be added to a liquid, such as an animal's drinking water.

As used herein, the term "contacted" refers to the indirect or direct application of a Bacillus-based ingredient described herein to a product (e.g., feed).

Examples of application methods that may be used include, but are not limited to, treating the product with a material containing a Bacillus-based component, direct application by mixing the feed additive composition Bacillus-based component described herein with the product, spraying such a feed additive composition onto the product surface, or immersing the product in a feed additive composition preparation. In one embodiment, the feed additive composition Bacillus-based component described herein is preferably mixed with the product (e.g., feed). Alternatively, the feed additive composition may be included in the feed emulsion or ingredients, allowing the composition to impart a performance benefit.

The methods of preparing the Bacillus-based ingredients described herein may also include the further step of pelletizing the powder. The powder may be mixed with other ingredients known in the art. The powder, or a mixture containing the powder, may be extruded through a die and the resulting strands cut into suitable pellets of variable length.

Optionally, the pelletization process may include a steam treatment or conditioning step prior to the formation of pellets. The mixture containing the powder may be placed in a conditioner (e.g., a mixer with steam injection). The mixture is heated in the conditioner to a specific temperature such as 60-100°C, with typical temperatures being 70°C, 80°C, 85°C, 90°C, or 95°C. Residence times may vary from a few seconds to a few minutes or even hours. For example, 5 seconds, 10 seconds, 15 seconds, 30 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, and 1 hour.

It will be understood by those skilled in the art that different animals have different feed requirements, and that the same animal may have different feed requirements depending on the purpose for which the animal is being raised.

Optionally, the feed may also include additional minerals, such as calcium, and/or additional vitamins. In some embodiments, the feed is a corn soybean meal mix.

Feed is typically produced in a feed mill, where the raw material is first ground to a suitable particle size and then mixed with appropriate additives. The feed may then be produced as a mash or pellets; the latter typically involves raising the temperature to a target level and then passing the feed through a die to produce pellets of a particular size. The pellets are cooled. Liquid additives such as fats and enzymes may then be added. Feed production may also include further steps including extrusion or expansion prior to pelleting, particularly by suitable techniques which may include at least the use of steam.

As mentioned above, the Bacillus-based ingredients and/or feeds comprising same may be used in any suitable form. The Bacillus-based ingredients and/or feeds comprising same may be used in the form of solid or liquid preparations or alternatives thereof. Examples of solid preparations include powders, pastes, boli, capsules, pellets, tablets, dusts, and granules, which may be soluble, spray dried, or freeze dried. Examples of liquid preparations include, but are not limited to, aqueous solutions, organic solutions or aqueous-organic solutions, suspensions, and emulsions.

In some applications, the feed additive composition may be mixed with the feed or administered in the drinking water.

A Bacillus-based ingredient comprising mixing and (optionally) packaging the Bacillus-based ingredient described herein with a feed-acceptable carrier, diluent, or additive.

The feed and/or Bacillus-based ingredient may be combined with at least one mineral and/or at least one vitamin. The composition thus obtained may be referred to herein as a premix. The feed may comprise at least 0.0001% by weight of the Bacillus-based ingredient. Suitably, the feed may comprise at least 0.0005% by weight; at least 0.0010% by weight; at least 0.0020% by weight; at least 0.0025% by weight; at least 0.0050% by weight; at least 0.0100% by weight; at least 0.020% by weight; at least 0.100% by weight; at least 0.200% by weight; at least 0.250% by weight; at least 0.500% by weight of the Bacillus-based ingredient.

Preferably, the food or Bacillus-based ingredient may further comprise at least one physiologically acceptable carrier, preferably selected from at least one of maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat or wheat components, sucrose, _starch , _Na2SO4 , talc, PVA, and mixtures thereof. In a further embodiment, the food or feed may further comprise a metal ion chelator, which may be selected from EDTA or citric acid.

In one embodiment, the Bacillus-based ingredients described herein (whether encapsulated or not) may be formulated with at least one physiologically acceptable carrier selected from at least one of the following: maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat or wheat components, sucrose, starch, Na ₂ SO ₄ , talc, PVA, sorbitol, benzoates, sorbates, glycerol, sucrose, propylene glycol, 1,3-propanediol, glucose, parabens, sodium chloride, citrates, acetates, phosphates, calcium, metabisulfites, formates, and mixtures thereof.

In some embodiments, the Bacillus-based components described herein will be present in a physiologically acceptable carrier. Suitable carriers can be large, slowly metabolized macromolecules (e.g., proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles). Pharmaceutically acceptable salts (e.g., mineral acid salts such as hydrochlorides, hydrobromides, phosphates, and sulfates, or salts of organic acids such as acetates, propionates, malonates, and benzoates) can be used. Pharmaceutically acceptable carriers in therapeutic compositions can further include liquids such as water, saline, glycerol, and ethanol. In addition, auxiliary substances such as wetting agents, emulsifying agents, or pH buffering substances can be present in such compositions. Such carriers allow the pharmaceutical compositions to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, and suspensions for ingestion by the patient. Once formulated, they can be administered directly to the subject. The subject to be treated may be an animal.

The compositions disclosed herein may provide one or more performance benefits (i.e., animal performance) in an animal, the performance benefits being believed to be selected from the group consisting of increased weight gain, increased feed rate, improved gut barrier integrity, reduced mortality, and reduced fecal shedding of E. coli.

Preferably, "animal performance" is determined by feed efficiency, and/or animal weight gain, and/or feed conversion ratio.

"Improved animal performance" means increased feed efficiency, and/or increased weight gain, and/or reduced feed conversion ratio, and/or improved gut barrier integrity, and/or reduced mortality, and/or reduced fecal shedding of E. coli, compared to a feed or feed additive composition not comprising the composition disclosed herein.

Preferably, "improved animal performance" means increased feed efficiency and/or increased weight gain and/or decreased feed conversion ratio. As used herein, the term "feed efficiency" refers to the amount of weight gain of an animal that occurs when the animal is fed an adequate amount of food or a specified amount of food over a period of time.

"Increased feed efficiency" means an increase in weight gain per unit of feed intake due to the use of the feed additive composition of the present invention in a feed, compared to animals fed without the presence of said feed additive composition.

As used herein, the term "feed conversion ratio" refers to the amount of feed fed to an animal to increase the animal's body weight by a specified amount.

Improved feed conversion means lower feed conversion ratios.

"Lower feed conversion ratio" or "improved feed conversion ratio" means that use of the feed additive composition in a feed reduces the amount of feed that needs to be fed to an animal to gain a specified amount of weight compared to the amount of feed that would be required to gain the same amount of weight if the feed did not contain the feed additive composition disclosed herein.

Gut integrity and microbiota appear to help maintain gut health. Supporting the intestinal barrier helps reduce the risk of infection and inflammation. For example, tight junctions are closely associated regions of two cells whose membranes join together to form a barrier that is substantially impermeable to fluids. The ability to protect the integrity of tight junctions can improve the health of animals. Tight junctions also need to be maintained to avoid "leaking gut," which can lead to further cell damage. It is therefore desirable to improve gut integrity by supporting or increasing an animal's ability to maintain a well-regulated barrier function that prevents unintended entry of bacteria into the animal's body.

Tight junctions (also known as occluding junctions) are closely associated regions of two cells whose membranes join together to form a barrier that is substantially impermeable to fluids. In terms of gut health, tight junctions are channels between intestinal epithelial cells that can lead to either good gut integrity or poor gut integrity. When good gut integrity is present, tight junction proteins such as claudin 3 and occludin are expressed at higher levels between two epithelial cells and help provide a barrier (reduced permeability) that can prevent the translocation of pathogens from the gut lumen into the systemic circulation. Claudins are the most important family of tight junction proteins, and claudin 3 is one of the genes that encode these tight junction proteins. Occludin is another important tight junction protein. Claudins and occludin were the first tight junction integral membrane proteins identified. Measurement of tight junction protein RNA can serve as an indicator of barrier integrity and gut health, since if tight junctions are not maintained in an animal's digestive tract, permeability can occur that can allow translocation of pathogens and toxins from the intestinal lumen into the systemic circulation, thus compromising the animal's health or even resulting in the death of the animal (e.g., sepsis).

Reduction in fecal shedding of E. coli through use of the compositions taught herein may reduce the further spread of E. coli based infections, as well as improve animal performance.

Nutrient digestibility, as used herein, refers to the percentage of a nutrient that is lost from the digestive tract or a particular segment of the digestive tract (e.g., the small intestine). Nutrient digestibility may be measured as the difference between what is administered to a subject and what is passed into the subject's feces, or it may be measured as the difference between what is administered to a subject and what remains in the digestive tract contents of a particular segment of the digestive tract (e.g., the ileum).

Nutrient digestibility, as used herein, may be measured by the difference between the intake of a nutrient and the nutrient excreted by the total amount of waste recovered during a period of time; or by the use of an inert marker that allows the researcher to calculate the amount of nutrient not absorbed by the animal and lost in the entire digestive tract or in a section of the digestive tract. Such inert markers may be titanium dioxide, chromium oxide, or acid-insoluble ash. Digestibility may be expressed as a percentage of the nutrient in the feed or as mass units of digestible nutrient per mass unit of nutrient in the feed.

Nutrient digestibility, as used herein, includes starch digestibility, fat digestibility, protein digestibility, and amino acid digestibility.

Energy digestibility, as used herein, means the total energy of the feed consumed minus the total energy of the feces or the total energy of the feed consumed minus the total energy of the digestive contents remaining in a particular section of the animal's digestive tract (e.g., the ileum). Metabolic energy, as used herein, refers to apparent metabolic energy and means the total energy of the feed consumed minus the total energy contained in the feces, urine, and gaseous products of digestion. Energy digestibility and metabolizable energy may be measured as the difference between total energy intake and total energy excreted in the feces or in the digestive contents present in a particular section of the digestive tract, using the same methods as for measuring digestibility of nutrients, with appropriate correction for nitrogen excretion to calculate the metabolic energy of the feed.

In some embodiments, the compositions described herein may improve the digestibility or utilization of dietary hemicellulose or fiber in a subject.

The term survival rate, as used herein, refers to the number of subjects who are surviving. The term "improved survival rate" may be interchangeably referred to as "reduced mortality rate."

"Increased weight gain" refers to the weight gain of an animal when fed a feed containing the feed additive composition compared to an animal fed a feed without the feed additive composition present.

Non-limiting examples of the compositions and methods disclosed herein include:
1. A composition for preventing and/or treating E. coli based infections in animals, comprising a Bacillus-based direct-fed microbial component comprising Bacillus strain 3BP5 (NRRL B-50510); B. amyloliquefaciens strain 918 (NRRL B-50508) and B. amyloliquefaciens strain 15AP4 (PTA-6507), alone or in combination with culture supernatants derived from these strains.
2. The composition of embodiment 1, wherein the composition provides one or more performance benefits selected from the group consisting of increased weight gain, increased feed rate, improved gut barrier integrity, reduced mortality, and reduced shedding of E. coli in the feces.
3. The composition of embodiment 1 or 2, wherein the direct-fed microorganism is in the form of an endospore.
4. The composition according to embodiment 1 or 2, wherein the composition further comprises at least one enzyme, which may optionally be encapsulated.
5. The composition of embodiment 3, wherein the composition further comprises at least one enzyme, which may optionally be encapsulated.
6. The composition of embodiment 4, wherein the at least one enzyme is selected from the group consisting of phytase, protease, amylase, xylanase, and beta-glucanase.
7. The composition of any one of embodiments 1, 2, 5, or 6, wherein the composition is a feed additive composition or a premix.
8. The composition of embodiment 3, wherein the composition is a feed additive composition or a premix.
9. The composition of embodiment 4, wherein the composition is a feed additive composition or a premix.
10. A feed comprising the feed additive composition according to embodiment 7.
11. A feed comprising the feed additive composition according to embodiment 8 or 9.
12. A kit comprising the feed additive composition of embodiment 7 and instructions for administration.
13. A kit comprising the feed additive composition of embodiment 8 or 9 and instructions for administration.
14. A method for preventing and/or treating an E. coli based infection in an animal comprising administering an effective amount of a composition comprising a Bacillus-based direct-fed microbial component, including Bacillus subtilis strain 3BP5 (NRRL B-50510); Bacillus amyloliquefaciens strain 918 (NRRL B-50508) and strain 15AP4 (PTA-6507).
15. The method of embodiment 14, wherein the composition provides one or more performance benefits selected from the group consisting of increased weight gain, increased feed rate, improved gut barrier integrity, and reduced fecal shedding of E. coli.
16. The method of embodiment 14 or 15, wherein the direct-fed microorganism is in the form of an endospore.
17. The method of embodiment 14 or 15, wherein the composition further comprises at least one enzyme, which may optionally be encapsulated.
18. The method of embodiment 16, wherein the composition further comprises at least one enzyme, which may optionally be encapsulated.
19. The method of embodiment 17, wherein the at least one enzyme is selected from the group consisting of phytase, protease, amylase, xylanase, and beta-glucanase.
20. The method of embodiment 18, wherein the at least one enzyme is selected from the group consisting of phytase, protease, amylase, xylanase, and beta-glucanase.
21. The method of any one of embodiments 14, 15, 19, and 20, wherein the composition is a feed additive composition or a premix.
22. The method of embodiment 16, wherein the composition is a feed additive composition or a premix.

Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, N.Y. (1991) provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

The present disclosure is further defined in the following examples. It should be understood that the examples, while illustrating certain embodiments, are given for illustrative purposes only. From the above discussion and examples, one skilled in the art can ascertain the essential features of the present disclosure and can make various changes and modifications to suit various applications and conditions without departing from the spirit and scope of the present invention.

Example 1
Effects of three strains of Bacillus-based direct-fed microorganisms (Bacillus 3BP5, 918, 15AP4) on growth performance, fecal E. coli shedding score, and intestinal barrier function of weaned pigs challenged with F18 enterotoxigenic Escherichia coli Materials and Methods Husbandry and Environment The use of animals and experimental protocols were approved by the Animal Experiment Committee. The basal diet is formulated at the time of feeding to be balanced for energy and protein and to meet or exceed the nutrient requirements of growing pigs of this age as recommended by the NRC (2012) (Table 1).

This basal ration is divided and then treated with direct-fed microorganisms (DFM) as shown in Table 1. During feed mixing, the mixer is flushed to prevent cross-contamination of the rations. Samples are taken from each treatment ration at the beginning, middle, and end of each batch, blended together, and the DFM in the feed is enumerated. Samples from each treatment ration are taken during mixing and stored at -20°C until needed.

Experimental Design A total of 72 newly weaned mixed sex (50% barrows and gilts) pigs weighing 6.44±0.64 kg are used in the 17-day study. Each pig is genetically tested to ensure susceptibility to F18 E. coli. The pigs are individually weighed, blocked by weight, and randomly assigned within blocks to 1-4 regular feed treatments. The pigs are housed 2 pigs per pen and kept in one of two separate rooms based on their challenge status: a smaller room for 18 non-challenged control pigs (9 pens; PC treatment) and another larger room for 54 challenged pigs (27 pens; 9 pens per treatment for NC, NC+DFM1, and NC+DFM2). All pigs are kept in environmentally controlled rooms. One side of each pen is equipped with a stainless steel automatic feeder and nipple waterer that allows the pigs free access to feed and water. The experiment was carried out over 17 days. It consisted of a 7-day adaptation period to the regular diet followed by a 10-day challenge period. On day 7 after weaning (day 7 of the study), pigs in the NC, NC+DFM1, and NC+DFM2 treatment groups are orally infected with F18 enterotoxigenic E. coli (ETEC). The PC treatment group is not challenged with E. coli.

Growth performance and collection and analysis of fecal samples Body weight and feed consumption are measured on days 0, 7, and 17 (end of study) to monitor average daily gain (ADG), average daily feed intake (ADFI), and gain to feed ratio (G:F). Fecal swabs are collected on day 7 (just before challenge) and on days 1, 2, 3, 4, 5, 6, 7, and 10 post infection (dpi) to assess E. coli shedding. Fecal scores are visually ranked daily in a blinded fashion using the following scale: 1 = solid; 2 = semi-solid; 3 = semi-liquid; 4 = liquid.

On dpi 10 (the final day of the study), one pig from each pen will be euthanized by capture bolt and subsequently exsanguinated to harvest the following tissues: freshly fixed ileal and colonic segments, and ileal and colonic gut contents. Ileal tissue segments will be analyzed by PCR to determine RNA expression of tight junction proteins that affect tight junction (intestinal) integrity.

Growth performance data will be analyzed using PROC MIXED (SAS 9.4) with initial body weight as a covariate. Time course data will be analyzed as repeated measures in PROC GLIM MIX.

Results Growth performance: There were no significant differences (P>0.10) in ADG, ADFI, or G:F among the four treatments during the 7-day adaptation period. However, weight gain with NC+DFM2 was numerically greater than all other treatments. Pigs on the NC+DFM2 regular diet gained 0.781 kg (final weight-initial weight) during the first 7 days of the study, while PC, NC, and NC+DFM1 gained 0.239, 0.188, and 0.365 kg, respectively (Table 3).

This means that pigs fed the NC+DFM2 diet also had numerically higher ADG and G:F compared to all other treatments during the first 7 days of diet adaptation (Table 3).

After the 10-day challenge period (days 8-17 of the study), pigs treated with NC, NC+DFM1, and NC+DFM2 had lower final BW (P=0.002), lower ADG (P<0.0001), and lower ADFI (P=0.002) compared to PC (Table 4). This is the expected outcome of a successful E. coli challenge. However, only the NC and NC+DFM1 treatment groups had lower G:F compared to the control.

NC+DFM2 pigs had a median G:F that was 19% higher than NC (0.647 vs. 0.542) and statistically similar to PC (Table 4).

Pigs fed DFM2 gained 2.477 kg over the 10-day challenge period, whereas pigs fed NC or NC + DFM1 gained only 2.312 and 1.881 kg, respectively (Table 4).

E. coli shedding scores: Overall, PC-treated E. coli shedding scores (SS) were lower compared to challenge treatments, as expected (P<0.01, Table 6). NC+DFM2 treatment increased SS at 2 dpi (P=0.04) but decreased SS at 7 dpi (P=0.003) compared to NC (Table 5). NC+DFM2 numerically decreased SS relative to NC at 5 and 10 dpi (Table 5).

Overall, NC+DFM1 and NC+DFM2 numerically reduced E. coli fecal shedding compared to NC (1.96 and 2.03 versus 2.12, respectively; Table 6).

Fecal scores: From 3 days post-infection to 8 dpi, PCs had lower fecal scores (FS) compared to NCs, as expected (P<.0001, Table 7). At 2 dpi, pigs fed NC+DFM2 had higher FS compared to NC pigs, but at 7 dpi, DFM2 pigs had lower FS compared to NC pigs (P<0.10; Table 7).

Similarly, pigs fed DFM2 had numerically lower FC compared to pigs fed NC on dpi 4-10. DFM1 did not significantly reduce FS compared to NC on any dpi (Table 7).

Tight Junction Protein RNA Expression (Intestinal Integrity):
Tight junctions (also known as occluding junctions) are closely associated regions of two cells whose membranes join together to form a barrier that is substantially impermeable to fluids. In terms of gut health, tight junctions are channels between intestinal epithelial cells that can lead to either good gut integrity or poor gut integrity. In the presence of good gut integrity, tight junction proteins such as claudin 3 and occludin are expressed between two epithelial cells and help provide a barrier that can prevent the translocation of pathogens from the gut lumen to the systemic circulation. Claudins are the most important family of tight junction proteins, and claudin 3 is one of the genes that code for these tight junction proteins. Occludin is another important tight junction protein. Claudins and occludin were the first tight junction integral membrane proteins identified. Measurement of tight junction protein RNA can serve as an indicator of barrier integrity and intestinal health, since if tight junctions are not maintained in an animal's digestive tract, permeability can occur that can allow translocation of pathogens and toxins from the intestinal lumen into the systemic circulation, thus affecting the animal's health or even resulting in the death of the animal.

In this experiment, the dietary treatment had no significant effect on RNA expression of the tight junction protein claudin-3; however, PC was found to be numerically higher than NC and NC+DFM1 (0.982 vs. 0.638 and 0.648, respectively; Figure 1). However, NC+DFM2 showed RNA expression of claudin-3 that was numerically similar to PC (0.945 vs. 0.982; Figure 1).

Treatment (i.e., DFM1 or DFM2) had a significant effect on RNA expression of the tight junction protein occludin, such that NC and NC+DFM1 had lower occludin expression compared to PC, while NC+DFM2 was intermediate between PC and NC (P=0.045). P values indicate overall differences between treatments as shown in Figure 1.

Claims (15)

1. A composition for preventing and/or treating an E. coli based infection in an animal, comprising:
Bacillus-based direct-fed microbial components include Bacillus subtilis 3BP5 ( NRRL B-50510) ; Bacillus amyloliquefaciens 918 (NRRL B-50508) and 15AP4 (PTA-6507), either alone or in combination with culture supernatants derived from these strains;
The composition provides one or more performance benefits selected from the group consisting of increased weight gain, increased feed rate, improved intestinal barrier integrity, reduced mortality, and reduced shedding of E. coli in the feces. The composition of claim 1, wherein the directly administered microorganism is in the form of an endospore. The composition according to claim 1 or 2, further comprising at least one enzyme. The composition of claim 3, wherein the at least one enzyme can be encapsulated. The composition according to claim 3 or 4, wherein the at least one enzyme is selected from the group consisting of phytase, protease, amylase, xylanase, and beta-glucanase. The composition according to any one of claims 1 to 5, wherein the composition is a feed additive composition. The composition according to any one of claims 1 to 5, wherein the composition is a premix. A feed containing the feed additive composition according to claim 6. A kit comprising the feed additive composition of claim 6 and instructions for administration. 1. A method for preventing and/or treating an E. coli based infectious disease in an animal (excluding humans), comprising:
administering an effective amount of a composition comprising a Bacillus-based direct-feed microbial component, including Bacillus subtilis 3BP5 ( NRRL B-50510) ; Bacillus amyloliquefaciens 918 (NRRL B-50508) and 15AP4 (PTA-6507) ;
The method, wherein the composition provides one or more performance benefits selected from the group consisting of increased weight gain, increased feed rate, improved intestinal barrier integrity, reduced mortality, and reduced E. coli shedding in the feces. The method of claim 10, wherein the directly administered microorganism is in the form of an endospore. The method of claim 10 or 11, wherein the composition further comprises at least one enzyme. The method of claim 12, wherein the at least one enzyme can be encapsulated. The method according to claim 12 or 13, wherein the at least one enzyme is selected from the group consisting of phytase, protease, amylase, xylanase, and beta-glucanase. The method according to any one of claims 10 to 14, wherein the composition is a feed additive composition or a premix.

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